Intermediate filaments (IF) are the ubiquitous constituents of the cytoskeletons of eukaryote cells. They consist of at least 6 different types, of which the most numerous and complex are the type I and type II keratins that are widely expressed in epithelia. We are interested in not only the structure, function and expression of keratin IF of human skin and their roles in keratinopathy diseases, but also of the related IF of other cell types in order to understand their roles in biology. Discovery of new mutations in keratinopathies We have discovered a variety of new mutations in some of the genes encoding keratin chains expressed in human epidermis. These occupy novel heretofore unreported residue positions near the beginning or end of the rod domain, and include: T109P of the 2B rod domain segment of the K2e gene in ichthyosis bullosa of Siemens; A12P of the 1A rod domain segment of the K10 gene in epidermolytic hyperkeratosis; and E106D of the 2B rod domain segment of the K1 gene in epidermolytic hyperkeratosis. Each of these mutations results in substitutions of inappropriate amino acids which result in defective keratin intermediate filaments in vivo (and hence result in clinical disease), and in filaments assembled in vitro. These data not only augment a growing database of keratin mutations, but also have provided useful clues on higher-order levels of structural information in intact filaments. Ongoing structural studies While the roles of the keratins in many genetic diseases are now well understood, further structural studies are necessary to develop rational approaches to therapy. We have initiated two types of structural/functional studies. In the first, we have developed synthetic peptides corresponding to the beginning of the 1A or end of the 2B rod domain segments. Several of these synthetic peptides have been injected into living cells to explore their dynamic behavior. Most function as very specific reagents for the disruption of all types of IF, but do not interfere with the state of assembly of microtubules or microfilaments. However, they do affect the supramolecular organization of them, thus indicating that all three components of the cytoskeleton function cooperatively in cells. On the other hand, we have discovered that an H1 peptide, a sequence which is specifically found only in type II keratin chains, is a very specific poison for the organization of keratin IF in epithelia. In those cultured cells which express both keratin IF and vimentin IF, only the keratin IF organization is disrupted. In a second experimental approach, we have also synthesized a series of peptides of sequence corresponding to keratin chains which are involved in the important overlap regions in assembled filaments, for biophysical structural studies, solution nmr, and for X-ray crystallography. To date, 2B peptides derived from the type III protein vimentin show simple dimerization, are essentially monodisperse in solution, and possess <90% a-helix, thus suggesting they will be useful to obtain detailed atomic-resolution structural information. The role of ionic interactions in IF structure The discovery that an E106D substitution causes disease has prompted us to re-evaluate the role of ionic interactions in the stability of the keratin chains in IF. We note that there are several potential pairs of charged residues which occupy e-f positions of the heptad repeat that have been precisely conserved in all IF chain types, including E106. Accordingly, using the type III vimentin and types I/II keratin 5/14 paradigms, we have expressed in bacteria a large number of mutant chains for use in in vitro assembly experiments to investigate the level of IF structural hierarchy at which the conserved charged pairs play important roles. The assays used will involve visual examination of the IF by negative staining, coiled-coil molecule stabilities as assessed by urea dissolution experiments, and isolation and characterization of a-helix-enriched proteolytic fragments to examine molecular alignments. Ongoing crosslinking studies In explorations of the structure and organization of the cornified cell envelope, we have discovered a large number of peptides involving crosslinks between a keratin chain and a variety of other envelope proteins. Notably, the vast majority of crosslinks involve a very specific and precisely conserved lysine residue located in the V1 region of the head domain of the type II keratins K1, K5 or K6. Interestingly, we have previously identified a case of non-epidermolytic palmaplantar keratoderma in which this conserved lysine residue of the K1 chain was substituted by isoleucine. Detailed ultrastructural analyses of the patient tissue at the level of the electron microscope revealed a severe discoordination between the cell periphery and the keratin IF cytoskeleton in the upper granular cells of the epidermis, proximal to the formation of the cornified cell envelope. Therefore, we believe this residue is critically involved in the structural organization of the cytoskeleton with the cornified cell envelope in terminally differentiated epidermis, and other related stratified squamous epithelia. Loss of this critical mode of organization results in a diminished barrier function for the epidermis. Future studies will involve attempts to ablate this lysine residue to create a mouse model for this disease and to further study the connection between cytoskeletal-cornified cell envelope coordination and barrier function. In addition, we have identified other crosslinks which reveal that the keratin IF are attached to the desmoplakin component of desmosomes indirectly through a series of related intermediate filament associated proteins. Further work will be directed to understand the complexity of these apparent connections and role in disease. The organization of molecules in various IF types We have previously demonstrated by detailed crosslinking experiments that pairs of epidermal keratin molecules are aligned in three basic modes termed A11, A22 and A12. When assimilated into IF, pairs of molecules in the same axial row adopt a fourth mode termed ACN, in which the end of one molecule overlaps the beginning of the adjacent molecule by about 1 nm. Interestingly, almost all known keratinopathy mutations/substitutions reside in this overlap 'window'.